Uses of Bcl-2 Antagonists for Treating Cancer and Diagnostics Related Thereto

ABSTRACT

In certain embodiments, this disclosure relates to method of treating and diagnosing cancer by administering a Bcl-2 inhibitor optionally in combination with a mitochondrial complex II inhibitor. In certain embodiments, a subject is diagnosed with, exhibiting symptoms of, or at risk of cancer wherein the cancer is a hematological malignancy such as multiple myeloma, leukemia, or lymphoma.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.62/663,104 filed Apr. 26, 2018. The entirety of this application ishereby incorporated by reference for all purposes.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under CA208328 awardedby the National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND

Overexpression of the Bcl-2 family members is commonly associated withtumor maintenance, progression, and chemoresistance. Inhibitors of Bcl-2family members promote apoptosis of cancer cells. Venetoclax(VENCLEXTA™) is a Bcl-2 antagonist used to treat chronic lymphocyticleukemia (CLL). Elmore et al. report methods of treatment usingselective Bcl-2 inhibitors. U.S. Pat. No. 9,345,702. See also WO2005/049593, and WO 2005/024636.

Multiple myeloma (MM) is a plasma cell malignancy. The development ofchemo-resistant MM is common, with approximately 20% of patientssuccumbing to aggressive treatment-refractory disease within a shortperiod of diagnosis. Resistance is primarily associated with evasion ofapoptosis connected to the inability to release sufficient pro-apoptoticBcl-2 proteins. Thus, finding improved therapeutic strategies areneeded.

A common translocation in multiple myeloma (MM) is t(11;14)(q13;q32).According to several studies, this translocation represents a uniquesubset of patients with worse than standard risk outcomes. Certainchromosomal aberrations in combination with t(11;14) alone are alsoreported. See Leiba et al. Genes Chromosomes Cancer. 2016, 55(9):710-8.About 40% of the t(11;14) exhibiting patients are responsive to singleagent venetoclax. Thus, there is a need to find improved diagnosticstrategies.

Bajpai et al. report targeting glutamine metabolism in multiple myelomaenhances BIM binding to Bcl-2 eliciting synthetic lethality tovenetoclax. Oncogene, 2016, 35(30):3955-64.

References cited herein are not an admission of prior art.

SUMMARY

In certain embodiments, this disclosure relates to method of treatingand diagnosing cancer by administering a Bcl-2 inhibitor optionally incombination with a mitochondrial complex II inhibitor. In certainembodiments, a subject is diagnosed with, exhibiting symptoms of, or atrisk of cancer wherein the cancer is a hematological malignancy such asmultiple myeloma, leukemia, or lymphoma.

In certain embodiments, this disclosure relates to methods of treatingcancer comprising administering an effective amount of a Bcl-2 inhibitorin combination with a mitochondrial complex II inhibitor to a subject inneed thereof. In certain embodiments, the mitochondrial complex IIinhibitor is thenoyltrifluoroacetone or atpenin A5. In certainembodiments, the Bcl-2 inhibitor is venetoclax or navitoclax orderivative thereof.

In certain embodiments, this disclosure relates to a method of treatingmultiple myeloma comprising administering an effective amount ofvenetoclax in combination with a thenoyltrifluoroacetone or atpenin A5to a subject in need thereof.

In certain embodiments, the subject is diagnosed with cancer orhematological malignancy. In certain embodiments, the hematologicalmalignancy is multiple myeloma, leukemia, or lymphoma. In certainembodiments, the hematological malignancy is acute lymphoblasticleukemia (ALL), acute myelogenous leukemia (AML), chronic lymphocyticleukemia (CLL), small lymphocytic lymphoma (SLL), chronic myelogenousleukemia, acute monocytic leukemia (AMOL), Hodgkin's lymphomas, andnon-Hodgkin's lymphomas such as Burkitt lymphoma, B-cell lymphoma.

In certain embodiments, this disclosure contemplates identifyingt(11;14) patients who will respond to venetoclax as well to identifyingstrategies to sensitize the broader resistant (t(11;14) and non t(11;14)myeloma population to venetoclax.

In certain embodiments, this disclosure relates to methods comprising,a) isolating a sample of cancer cells from a subject, b) mixing thesample with mitochondrial complex II, succinate, and ubiquinone; and c)measuring succinate ubiquinone reductase (SQR) activity providingmeasured SQR activity. In certain embodiments, the method furthercomprises diagnosing the subject as sensitive to or in need of achemotherapy treatment comprising administering a Bcl-2 inhibitor incombination with a mitochondrial complex II inhibitor, if the measuredSQR activity is higher than a control level. In certain embodiments, thesubject is or is not diagnosed with a t(11;14) translocation.

In certain embodiments, the method further comprises diagnosing thesubject as not in need of or would not benefit from a chemotherapycomprising administering Bcl-2 inhibitor in combination with amitochondrial complex II inhibitor, if the measured SQR activity islower than a control level.

In certain embodiments, the method further comprises the step ofrecording the measurements. In certain embodiments, the measurements arerecorded in an electronic format. In certain embodiments, the methodfurther comprises the step of recording the diagnosis. In certainembodiments, the diagnosis is recorded in an electronic format. Incertain embodiments, the method further comprises the step of reportingthe measurements or diagnosis to a medical professional, the subject, orrepresentative thereof. In certain embodiments, the method furthercomprises administering an effective amount of a combination therapy ofBcl-2 inhibitor in combination with a mitochondrial complex II inhibitorto the subject.

In certain embodiments, this disclosure relates to methods of diagnosingand treating multiple myeloma comprising, a) isolating a sample ofmyeloma cells from a subject, b) mixing the sample with mitochondrialcomplex II, succinate, and ubiquinone; and c) measuring succinateubiquinone reductase (SQR) activity providing measured SQR activity; andd) treating the subject with a Bcl-2 inhibitor in combination withmitochondrial complex II inhibitor if the measured SQR activity ishigher than a control level.

In certain embodiments, this disclosure relates to methods of diagnosingand treating multiple myeloma comprising, a) isolating a sample ofmyeloma cells from a subject, b) mixing the sample with mitochondrialcomplex II, succinate, and ubiquinone; and c) measuring succinateubiquinone reductase (SQR) activity providing measured SQR activity; andd) treating the subject with venetoclax in combination withthenoyltrifluoroacetone or atpenin A5 if the measured SQR activity ishigher than a control level.

In certain embodiments, administering an effective amount of a Bcl-2inhibitor in combination with a mitochondrial complex II inhibitor isfurther in combination with an additional chemotherapy agent. In certainembodiments, an additional chemotherapy agent or combination iscontemplated to be selected from abemaciclib, abiraterone acetate,methotrexate, paclitaxel, adriamycin, acalabrutinib, brentuximabvedotin, ado-trastuzumab emtansine, aflibercept, afatinib, netupitant,palonosetron, imiquimod, aldesleukin, alectinib, alemtuzumab, pemetrexeddisodium, copanlisib, melphalan, brigatinib, chlorambucil, amifostine,aminolevulinic acid, anastrozole, apalutamide, aprepitant, pamidronatedisodium, exemestane, nelarabine, arsenic trioxide, ofatumumab,atezolizumab, bevacizumab, avelumab, axicabtagene ciloleucel, axitinib,azacitidine, carmustine, belinostat, bendamustine, inotuzumabozogamicin, bevacizumab, bexarotene, bicalutamide, bleomycin,blinatumomab, bortezomib, bosutinib, brentuximab vedotin, brigatinib,busulfan, irinotecan, capecitabine, fluorouracil, carboplatin,carfilzomib, ceritinib, daunorubicin, cetuximab, cisplatin, cladribine,cyclophosphamide, clofarabine, cobimetinib, cabozantinib-S-malate,dactinomycin, crizotinib, ifosfamide, ramucirumab, cytarabine,dabrafenib, dacarbazine, decitabine, daratumumab, dasatinib,defibrotide, degarelix, denileukin diftitox, denosumab, dexamethasone,dexrazoxane, dinutuximab, docetaxel, doxorubicin, durvalumab,rasburicase, epirubicin, elotuzumab, oxaliplatin, eltrombopag olamine,enasidenib, enzalutamide, eribulin, vismodegib, erlotinib, etoposide,everolimus, raloxifene, toremifene, panobinostat, fulvestrant,letrozole, filgrastim, fludarabine, flutamide, pralatrexate,obinutuzumab, gefitinib, gemcitabine, gemtuzumab ozogamicin,glucarpidase, goserelin, propranolol, trastuzumab, topotecan,palbociclib, ibritumomab tiuxetan, ibrutinib, ponatinib, idarubicin,idelalisib, imatinib, talimogene laherparepvec, ipilimumab, romidepsin,ixabepilone, ixazomib, ruxolitinib, cabazitaxel, palifermin,pembrolizumab, ribociclib, ti sagenlecleucel, lanreotide, lapatinib,olaratumab, lenalidomide, lenvatinib, leucovorin, leuprolide, lomustine,trifluridine, olaparib, vincristine, procarbazine, mechlorethamine,megestrol, trametinib, temozolomide, methylnaltrexone bromide,midostaurin, mitomycin C, mitoxantrone, plerixafor, vinorelbine,necitumumab, neratinib, sorafenib, nilutamide, nilotinib, niraparib,nivolumab, tamoxifen, romiplostim, sonidegib, omacetaxine, pegaspargase,ondansetron, osimertinib, panitumumab, pazopanib, interferon alfa-2b,pertuzumab, pomalidomide, mercaptopurine, regorafenib, rituximab,rolapitant, rucaparib, siltuximab, sunitinib, thioguanine, temsirolimus,thalidomide, thiotepa, trabectedin, valrubicin, vandetanib, vinblastine,vemurafenib, vorinostat, zoledronic acid, or combinations thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows data of the SQR activity assay in KMS11 cells. Cells areresuspended in permeabilization buffer (10 mM KH2PO4 pH 7.8, 2 mM EDTAand 1 mg/ml BSA). 20 mM succinate is added as a substrate with orwithout 5 mM malonate (an SDH inhibitor) followed by a 10 min incubationat RT. SQR activity is determined by measuring electron transfer fromsuccinate to decylubiquinone and 2,6-dichlorophenolindophenol (DCPIP)that leads to a change in absorbance of DCPIP monitored at 600 nm for 10min.

FIG. 1B shows data indicating succinate ubiquinone reductase activitycorrelates with ABT-199 sensitivity in myeloma cell lines. Evaluation ofSQR activity in ABT-199 senistive and resistant myeloma cell linescorrelated with decrease in absorbance of DCIP (Results arerepresentative of 4 independent evaluations with the mean and SEMplotted in calculation of SQR activity in nmoles/min/μ1 demonstratinglower SQR activity in sensitive cells.

FIG. 2A shows data indicating inhibition of Complex II/SQR by TTFAsensitizes MINI lines and patient cells to ABT-199. CD38+ve MINI cellsfrom myeloma patient samples exhibit significant cytotoxicity uponcombinatorial treatment with TTFA (100 μM) and ABT-199 (0.5 μM) for 48hrs. Complex I inhibition with piercidin or SDHA inhibition with 3-NPA.L363, MNI.15 and KMS 11 cells were cultured in the presence or absenceof 0.SμM ABT-199 with or without either Complex I inhibitor piericidin(0.001 mM) or Complex II inhibitor 3NPA (1 mM) or TTFA (100 μM) for 24hrs. SQR activity evaluated in Patient #3 is low and comparable to othersensitive lines.

FIG. 2B shows data indicating normal cell populations contained withinthe bone marrow aspirate of patient sample #2 did not exhibitsignificant cytotoxicity with either ABT-199/TTFA or the combination.Synthetic lethality are not cytotoxic as a single agent in cell lines orprimary normal cells associated with a myeloma patient sample. In sum,these results indicate SQR regulates Bcl-2 dependence and ABT-199sensitivity and validate that SQR activity is predictive of ABT-199sensitivity and response in MM.

FIG. 3 shows data indicating the Complex II inhibitor atpenin A5sensitizes resistant cell lines.

FIG. 4 shows data Complex II inhibitor with TTFA sensitizes MM cells toABT-199 and is additive to Dex-induced death. KMS11, L363, RPMI-8226,MIVI.1S, U266 (ABT-199 resistant cell lines) and KMS12PE and OCIMYS(ABT-199 sensitive cell lines) were cultured in presence of SQRinhibitor TTFA (100 uM) or DEX (0.5 uM) either alone or in combinationwith ABT-199 (0.5 uM) for 24 hrs. Cell viability was assessed by AnnexinV-DAPI staining and Flow cytometry.

FIG. 5A shows data indicating mutation at Qp site in SDHC inhibits SQRactivity and sensitizes KMS11 to ABT-199. KMS11 cells exhibiting SDHCKOand overexpressing SDHCWT or SDHCR72C mutant cDNA, were cultured inpresence of SQR inhibitor TTFA (100 uM) for 24 hrs. Succinate UbiquinoneReductase activity was assessed as a function of DCPIP absorbance at 600nm.

FIG. 5B shows data on cells from FIG. 5A that were also treated or nottreated with ABT-199 (0.5 uM) for 24 hrs. Cell viability was assessed byAnnexin V-DAPI staining and Flow cytometry.

DETAILED DESCRIPTION

Before the present disclosure is described in greater detail, it is tobe understood that this disclosure is not limited to particularembodiments described, and as such may, of course, vary. It is also tobe understood that the terminology used herein is for the purpose ofdescribing particular embodiments only, and is not intended to belimiting, since the scope of the present disclosure will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this disclosure belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present disclosure, the preferredmethods and materials are now described.

All publications and patents cited in this specification are hereinincorporated by reference as if each individual publication or patentwere specifically and individually indicated to be incorporated byreference and are incorporated herein by reference to disclose anddescribe the methods and/or materials in connection with which thepublications are cited. The citation of any publication is for itsdisclosure prior to the filing date and should not be construed as anadmission that the present disclosure is not entitled to antedate suchpublication by virtue of prior disclosure. Further, the dates ofpublication provided could be different from the actual publicationdates that may need to be independently confirmed.

As will be apparent to those of skill in the art upon reading thisdisclosure, each of the individual embodiments described and illustratedherein has discrete components and features which may be readilyseparated from or combined with the features of any of the other severalembodiments without departing from the scope or spirit of the presentdisclosure. Any recited method can be carried out in the order of eventsrecited or in any other order that is logically possible.

Embodiments of the present disclosure will employ, unless otherwiseindicated, techniques of immunology, medicine, organic chemistry,biochemistry, molecular biology, pharmacology, physiology, and the like,which are within the skill of the art. Such techniques are explainedfully in the literature.

It must be noted that, as used in the specification and the appendedclaims, the singular forms “a,” “an,” and “the” include plural referentsunless the context clearly dictates otherwise. In this specification andin the claims that follow, reference will be made to a number of termsthat shall be defined to have the following meanings unless a contraryintention is apparent.

Prior to describing the various embodiments, the following definitionsare provided and should be used unless otherwise indicated.

To the extent that structures provided herein are compounds withtautomers by hydrogen migration, a skilled artisan would understand theformula to cover all tautomeric forms.

As used herein, “salts” refer to derivatives of the disclosed compoundswhere the parent compound is modified making acid or base salts thereof.Examples of salts include, but are not limited to, mineral or organicacid salts of basic residues such as amines, alkylamines, ordialkylamines; alkali or organic salts of acidic residues such ascarboxylic acids; and the like. In preferred embodiment, the salts areconventional nontoxic pharmaceutically acceptable salts including thequaternary ammonium salts of the parent compound formed, and non-toxicinorganic or organic acids. Preferred salts include those derived frominorganic acids such as hydrochloric, hydrobromic, sulfuric, sulfamic,phosphoric, nitric and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, pamoic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicylic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isethionic, and the like.

“Subject” refers any animal, preferably a human patient, livestock, ordomestic pet.

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

“Cancer” refers any of various cellular diseases with malignantneoplasms characterized by the proliferation of cells. It is notintended that the diseased cells must actually invade surrounding tissueand metastasize to new body sites. Cancer can involve any tissue of thebody and have many different forms in each body area. Within the contextof certain embodiments, whether “cancer is reduced” may be identified bya variety of diagnostic manners known to one skill in the art including,but not limited to, observation the reduction in size or number of tumormasses or if an increase of apoptosis of cancer cells observed, e.g., ifmore than a 5% increase in apoptosis of cancer cells is observed for asample compound compared to a control without the compound. It may alsobe identified by a change in relevant biomarker or gene expressionprofile, such as PSA for prostate cancer, HER2 for breast cancer, orothers.

The term “prodrug” refers to an agent that is converted into abiologically active form in vivo. Prodrugs are often useful because, insome situations, they may be easier to administer than the parentcompound. They may, for instance, be bioavailable by oral administrationwhereas the parent compound is not. The prodrug may also have improvedsolubility in pharmaceutical compositions over the parent drug. Aprodrug may be converted into the parent drug by various mechanisms,including enzymatic processes and metabolic hydrolysis.

As used herein, the term “derivative” refers to a structurally similarcompound that retains sufficient functional attributes of the identifiedanalogue. The derivative may be structurally similar because it islacking one or more atoms, substituted, a salt, in differenthydration/oxidation states, or because one or more atoms within themolecule are switched, such as, but not limited to, replacing an oxygenatom with a sulfur or nitrogen atom or replacing an amino group with ahydroxyl group or vice versa. The derivative may be a prodrug.Derivatives may be prepare by any variety of synthetic methods orappropriate adaptations presented in synthetic or organic chemistry textbooks, such as those provide in March's Advanced Organic Chemistry:Reactions, Mechanisms, and Structure, Wiley, 6th Edition (2007) MichaelB. Smith or Domino Reactions in Organic Synthesis, Wiley (2006) Lutz F.Tietze hereby incorporated by reference.

The term “substituted” refers to a molecule wherein at least onehydrogen atom is replaced with a substituent. When substituted, one ormore of the groups are “substituents” or “radicals.” The molecule may bemultiply substituted. In the case of an oxo substituent (“═O”), twohydrogen atoms are replaced. Example substituents within this contextmay include halogen, hydroxy, alkyl, alkoxy, nitro, cyano, oxo,carbocyclyl, carbocycloalkyl, heterocarbocyclyl, heterocarbocycloalkyl,aryl, arylalkyl, heteroaryl, heteroarylalkyl, —NRaRb, —NRaC(═O)Rb,—NRaC(═O)NRaNRb, —NRaC(═O)ORb, —NRaSO2Rb, —C(═O)Ra, —C(═O)ORa,—C(═O)NRaRb, —OC(═O)NRaRb, —ORa, —SRa, —SORa, —S(═O)₂Ra, —OS(═O)₂Ra and—S(═O)₂ORa. Ra and Rb in this context may be the same or different andindependently hydrogen, halogen hydroxyl, alkyl, alkoxy, alkyl, amino,alkylamino, dialkylamino, carbocyclyl, carbocycloalkyl,heterocarbocyclyl, heterocarbocycloalkyl, aryl, arylalkyl, heteroaryl,and heteroarylalkyl.

If a substituent is described as being “substituted”, a non-hydrogenradical is in the place of hydrogen radical on a carbon or nitrogen ofthe substituent. Thus, for example, a substituted alkyl substituent isan alkyl substituent in which at least one non-hydrogen radical is inthe place of a hydrogen radical on the alkyl substituent. To illustrate,monofluoroalkyl is alkyl substituted with a fluoro radical, anddifluoroalkyl is alkyl substituted with two fluoro radicals. It shouldbe recognized that if there are more than one substitution on a substituent, each non-hydrogen radical may be identical or different(unless otherwise stated).

If a substituent is described as being “optionally substituted”, thesubstituent may be either (1) not substituted or (2) substituted. If asubstituent is described as being optionally substituted with up to aparticular number of non-hydrogen radicals, that substituent may beeither (1) not substituted; or (2) substituted by up to that particularnumber of non-hydrogen radicals or by up to the maximum number ofsubstitutable positions on the substituent, whichever is less. Thus, forexample, if a substituent is described as a heteroaryl optionallysubstituted with up to 3 non-hydrogen radicals, then any heteroaryl withless than 3 substitutable positions would be optionally substituted byup to only as many non-hydrogen radicals as the heteroaryl hassubstitutable positions. To illustrate, tetrazolyl (which has only onesubstitutable position) would be optionally substituted with up to onenon-hydrogen radical. To illustrate further, if an amino nitrogen isdescribed as being optionally substituted with up to 2 non-hydrogenradicals, then a primary amino nitrogen will be optionally substitutedwith up to 2 non-hydrogen radicals, whereas a secondary amino nitrogenwill be optionally substituted with up to only 1 non-hydrogen radical.

As used herein, “mitochondrial complex II” refers to succinatedehydrogenase that is a membrane complex in the Krebs cycle(tricarboxylic acid cycle). Mitochondrial complex II catalyzes theoxidation of succinate to fumarate and transfers the released electronsto ubiquinone, referred to as succinate ubiquinone reductase (SQR)activity. Succinate oxidation is coupled to reduction of ubiquinone toubiquinol at the mitochondrial inner membrane as one part of therespiration electron transfer chain. Mitochondrial complex II issometimes referred to as mitochondrial succinate:ubiquinoneoxidoreductase (mitochondrial SQR).

“Thenoyltrifluoroacetone” refers to a compound with the chemical name:4,4,4-trifluoro-1-(thiophen-2-yl)butane-1,3-dione or salts thereof, withthe chemical structure below, also referred to as TTFA.

As used herein, “venetoclax” refers to a compound with the chemicalname:4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamideand salts thereof, with the chemical structure below, also referred toas ABT-199.

In certain embodiments, derivatives of venetoclax include the followingcompounds:

4-[4-[(4′-chloro[1,1′-biphenyl]-2-yl)methyl]-1-piperazinyl]-N-[[4-[[(1R)-3-(dimethylamino)-1-[(phenylthio)methyl]propyl]amino]-3-nitrophenyl]suifonyl]-benzamide

4-(4-{[2-(4-Chlorophenyl)-5,5-dimethyl-1-cyclohexen-1-yl]methyl}-1-piperazinyl)-N-[(4-{[(2R)-4-(4-morpholinyl)-1-(phenylsulfanyl)-2-butanyl]amino}-3-[(trifluoromethyl)sulfonyl]phenyl)sulfonyl]benzamide

As used herein, the terms “prevent” and “preventing” include theprevention of the recurrence, spread or onset. It is not intended thatthe present disclosure be limited to complete prevention. In someembodiments, the onset is delayed, or the severity of the disease isreduced.

As used herein, the terms “treat” and “treating” are not limited to thecase where the subject (e.g. patient) is cured and the disease iseradicated. Rather, embodiments, of the present disclosure alsocontemplate treatment that merely reduces symptoms, and/or delaysdisease progression.

As used herein, the term “combination with” when used to describeadministration with an additional treatment means that the agent may beadministered prior to, together with, or after the additional treatment,or a combination thereof.

“Sample” or “biological sample” means biological material isolated froma subject. The biological sample may contain any biological materialsuitable for detecting the desired biomarkers, and may comprise cellularand/or non-cellular material from the subject. The sample can beisolated from any suitable biological tissue or fluid such as, forexample, prostate tissue, blood, blood plasma, urine, or cerebral spinalfluid (CSF).

Although some assay formats will allow testing of peripheral biologicalfluid samples without prior processing of the sample, it is typical thatperipheral biological fluid samples will be processed prior to testing.Processing generally takes the form of elimination of cells, such asplatelets in blood samples, and may also include the elimination ofcertain proteins, such as certain clotting cascade proteins from blood.In some examples, the peripheral biological fluid sample is collected ina container comprising EDTA.

The process of comparing a measured value and control, e.g., normal or areference value can be carried out in any convenient manner appropriateto the type of measured value and reference value for the protein atissue. As discussed above, measuring can be performed using quantitativeor qualitative measurement techniques, and the mode of comparing ameasured value and a reference value can vary depending on themeasurement technology employed. For example, when a qualitativecalorimetric assay is used to measure protein levels, the levels may becompared by visually comparing the intensity of the colored reactionproduct, or by comparing data from densitometric or spectrometricmeasurements of the colored reaction product (e.g., comparing numericaldata or graphical data, such as bar charts, derived from the measuringdevice).

The process of comparing may be manual (such as visual inspection by thepractitioner of the method) or it may be automated. For example, anassay device (such as a luminometer for measuring chemiluminescentsignals) may include circuitry and software enabling it to compare ameasured value with a reference value. Alternately, a separate device(e.g., a digital computer) may be used to compare the measured value(s)and the reference value(s). Automated devices for comparison may includestored reference values for the protein(s) being measured, or they maycompare the measured value(s) with reference values that are derivedfrom contemporaneously measured reference samples.

In some embodiments, the methods of the disclosure utilize simple orbinary comparison between the measured level(s) and the referencelevel(s) (e.g., the comparison between a measured level and a referencelevel determines whether the measured level is higher or lower than thereference level).

As described herein, biological fluid samples may be measuredquantitatively (absolute values) or qualitatively (relative values). Incertain aspects of the disclosure, the comparison is performed todetermine the magnitude of the difference between the measured andreference values (e.g., comparing the fold or percentage differencebetween the measured value and the reference value).

Succinate Ubiquinone Reductase Activity Predicts Cancer Sensitivity toBcl-2 Family Inhibitors

Depriving cells of glutamine enhanced binding of BIM to Bcl-2 therebysensitizing myeloma cell lines and patient samples to the BH3 mimeticvenetoclax (ABT-199), a highly selective, potent Bcl-2 antagonist.ABT-199 is however effective only in 40% of MM patients exhibiting the(11;14) translocation, warranting investigation of strategies toincrease the applicability of ABT-199. Tests were performed to determinewhether there were metabolic differences in ABT-199 sensitive t(11;14)and resistant cells that could inform us of a) predictors ofsensitivity; and b) metabolic targets that could be inhibited tosensitize resistant cells to ABT-199. Given that, tumor cellsdifferentially rely on glycolysis and mitochondrial oxidativephosphorylation oxygen consumption rates (OCR) were evaluated in ABT-199sensitive and resistant MM lines. ABT-199 sensitive lines exhibitedlower OCR in contrast to the resistant lines. Metabolite profiling andisotope tracing flux analyses of glutamine deprived myeloma cell linesrevealed specific reduction of TCA cycle metabolites includingsuccinate. TCA cycle intermediates are linked to mitochondrialrespiration via electron transport chain (ETC) complexes (I-V). Amongthe five ETC complexes, only Complex II/succinate dehydrogenase (SDH) isdirectly connected to the TCA cycle and ETC. SDH facilitates theoxidation of succinate to fumarate in the TCA cycle through its subunitSDHA, and transfers the released electrons to ubiquinone via its SDHB, Cand D subunits supporting the succinate ubiquinone reductase (SQR)activity of Complex II. Whether succinate dehydrogenase facilitatesABT-199 sensitization was investigated.

SDH ubiquinone reductase activity was evaluated in ABT-199 sensitive andresistant myeloma lines. ABT-199 sensitive cell lines had significantlylower SQR activity. Furthermore, inhibition of the SDH ubiquinonereductase with the Qp site inhibitor thenoyltrifluoroacetone (TTFA)sensitized resistant MM cells to ABT-199. Inhibition of Complex I withpiericidin or the SDHA subunit with 3-NPA were not as effective insensitizing MINI to ABT-199. Protein expression andco-immunoprecipitation analyses demonstrated an induction of BIM andenhanced BIM-Bcl-2 binding upon SDH inhibition with TTFA. TTFA treatedcells exhibited a reduction of Foxo3a phosphorylation and concomitantnuclear localization upon SDH inhibition suggestive of FOXO3a playing arole in BIM induction. TTFA+ABT-199 co-treatment induced apoptosis inABT-199 resistant myeloma patient cells without impacting viability ofthe associated normal populations. Thus, a tumor selective approach ofmetabolically-driven synthetic lethality relies on inhibition of themitochondrial enzyme SDH and use of the Bcl-2 antagonist, e.g., ABT-199for myeloma therapy. Experiments suggest SDH activity predicts ABT-199sensitivity and can be interrogated in MINI patients as a predictivemarker of ABT-199 sensitivity particularly within t(11;14) patientscurrently being enrolled for ABT-199 treatment.

Experiments indicate that ABT-199 sensitive myeloma cell lines exhibitreduced basal mitochondrial respiration. Complex II-succinate ubiquinonereductase activity is reduced in ABT-199 sensitive myeloma cell linesand patient samples. Inhibition of Complex II at the ubiquinone bindingsite potently sensitizes resistant myeloma cells to ABT-199.Pro-apoptotic Bcl-2 family protein BIM, is required to sensitizeglutamine-deprived or TTFA treated MM cells to ABT-199. Low succinateubiquinone reductase activity correlates with venetoclax sensitivity inmultiple myeloma.

Mitochondrial Complex II Inhibitor

Methods for identifying mitochondrial complex II inhibitors is found inJones et al. A spectrophotometric coupled enzyme assay to measure theactivity of succinate dehydrogenase. Anal Biochem, 2013, 442(1):19-23.In certain embodiments, the mitochondrial complex II inhibitor is any ofthe compounds disclosed herein or derivative thereof, e.g., compounddisclosed herein optionally substituted with one or more substituent.

In certain embodiments, the mitochondrial complex II inhibitor is TTFA[4,4,4-trifluoro-1-(2-thienyl)-1,3-butanedione], atpenin A5(3-[5,6-dichloro-2,4-dimethylhexanoyl]-4-hydroxy-5, 6-dimethoxy-1H-pyridin-2-one), atpenin A4(3-[5-chloro-2,4-dimethylhexanoyl]-2-hydroxy-5,6-dimethoxy-1H-pyridin-4-one), thiabendazole, flutolanil(N-(3-propan-2-yloxyphenyl)-2-(trifluoromethyl)benzamide),harzianopyridone(2-Hydroxy-5,6-dimethoxy-3-[(2R)-2-methylhex-4-enoyl]-1H-pyridin-4-one),1-(2,4-dihydroxy-5,6-dimethoxypyridin-3-yl)hexan-1-one, carboxin(5,6-dihydro-2-methyl-N-phenyl-1,4-oxathiin-3-carboxamide), and HQNO(2-heptyl-4-hydroxyquinoline N-oxide).

In certain embodiments, the mitochondrial complex II inhibitor is acompound of the following formula:

or salts thereof wherein R′ is heptyl, octyl, nonyl, decyl, or dodecyl.

In certain embodiments, the mitochondrial complex II inhibitor islonidamine (LND; 1-(2,4-dichlorobenzyl)-1H-indazole-3-carboxylic acid)or salts thereof.

Bcl-2 Inhibitors

In certain embodiments, the Bcl-2 inhibitor has the following formula:

or pharmaceutically acceptable salt thereof, wherein

A¹ is C(A²);

A² is H, F, Br, I, or Cl;

B¹ is R′, OR′, NUR′, NHC(O)R¹, F, Br, I, or Cl;

D¹ is H, F, Br, I, or Cl;

E¹ is H; and

Y¹ is H, CN, NO₂, F, Cl, Br, I, CF₃, OR¹⁷, SR¹⁷, SO₂R¹⁷, or C(O)NH₂;

R¹ is R⁴ or R⁵;

R⁴ is cycloalkyl or heterocycloalkyl;

R⁵ is alkyl or alkynyl, each of which is unsubstituted or substitutedwith one or two or three substituents independently selected from thegroup consisting of R⁷, OR⁷, NUR′, N(R⁷)₂, CN, OH, F, Cl, Br, and I;

R⁷ is R⁸, R⁹, R¹⁰, or R¹¹;

R⁸ is phenyl;

R⁹ is heteroaryl;

R¹⁰ is cycloalkyl, cycloalkenyl, or heterocycloalkyl; each of which isunfused or fused with R^(10A); R^(10A) is heteroarene;

R¹¹ is alkyl, which is unsubstituted or substituted with one or two orthree substituents independently selected from the group consisting ofR¹², OR¹², and CF₃;

R¹² is R¹⁴ or R¹⁶;

R¹⁴ is heteroaryl;

R¹⁶ is alkyl;

R¹⁷ is alkyl or alkynyl, each of which is unsubstituted or substitutedwith one or two or three substituents independently selected from thegroup consisting of R²², F, Cl, Br and I;

R²² is heterocycloalkyl;

wherein the cyclic moieties represented by R⁴, R⁸, R¹⁰, R²², areindependently unsubstituted or substituted with one or two or three orfour or five substituents independently selected from the groupconsisting of R^(57A), R²⁷, OR⁵⁷, SO₂R⁵⁷, C(O)R⁵⁷, C(O)OR⁵⁷,C(O)N(R⁵⁷)₂, NH₂, NHR⁵⁷, N(R⁵⁷)₂, NHC(O)R⁵⁷, NHS(O)₂R⁵⁷, OH, CN, (O), F,Cl, Br and I;

R^(57A) is spiroalkyl or spiroheteroalkyl;

R⁵⁷ is R⁵⁸, R⁶⁰, or R⁶¹;

R⁵⁸ is phenyl;

R⁶⁰ is cycloalkyl or heterocycloalkyl;

R⁶¹ is alkyl, which is unsubstituted or substituted with one or two orthree substituents independently selected from the group consisting ofR⁶², OR⁶², N(R⁶²)₂, C(O)OH, CN, F, Cl, Br, and I;

R⁶² is R⁶⁵ or R⁶⁶;

R⁶⁵ is cycloalkyl or heterocycloalkyl;

R⁶⁶ is alkyl, which is unsubstituted or substituted with OR⁶⁷;

R⁶⁷ is alkyl;

wherein the cyclic moieties represented by R^(57A), R⁵⁸, and R⁶⁰ areunsubstituted or substituted with one or two or three or foursubstituents independently selected from the group consisting of R⁶⁸, F,Cl, Br, and I;

R⁶⁸ is R⁷¹ or R⁷²;

R⁷¹ is heterocycloalkyl; and

R⁷² is alkyl, which is unsubstituted or substituted with one or two F.

In certain embodiments, the Bcl-2 inhibitor has the following formula:

Or salts thereof wherein

A¹ is N or CH;

B¹ is OR¹ or NHR¹;

Y¹ is CN, NO₂, CF₃, F or Cl;

R¹ is (CH₂)_(n)R²;

R² is cycloalkyl or heterocyclyl; wherein the heterocyclyl andcycloalkyl are optionally substituted with one or more independentlyselected R⁴, OR⁴, OH, CN, or F;

R³ is heteroaryl; wherein the heteroaryl is optionally substituted withone or more independently selected NH₂, Cl, or F;

R⁴ is alkyl, cycloalkyl, heterocyclyl, or spiroheterocyclyl; wherein thealkyl is optionally substituted with one or more F;

R⁵ is hydrogen or deuterium;

each R⁶ is independently selected from CH₃, spirocyclopropyl and OH;

m is 0, 1, 2, 3, 4, 5, or 6;

n is 0 or 1; and

p is 0, 1, or 2.

With regard to the various selective Bcl-2 inhibitor compoundsencompassed by the current disclosure, it should be understood thatvariable moieties herein are represented by identifiers (capital letterswith numerical and/or alphabetical superscripts) and may be specificallyembodied.

It is meant to be understood that proper valences are maintained for allmoieties and combinations thereof, that monovalent moieties having morethan one atom are drawn from left to right and are attached throughtheir left ends, and that divalent moieties are also drawn from left toright.

It is also meant to be understood that a specific embodiment of avariable moiety herein may be the same or different as another specificembodiment having the same identifier.

The term “alkyl” as used herein, means a straight or branched, saturatedhydrocarbon chain containing from 1 to 10 carbon atoms. The term “Cx-Cyalkyl” means a straight or branched chain, saturated hydrocarboncontaining x toy carbon atoms. For example “C₂-C₁₀ alkyl” means astraight or branched chain, saturated hydrocarbon containing 2 to 10carbon atoms. Examples of alkyl include, but are not limited to, methyl,ethyl, n-propyl, iso-propyl, n-butyl, sec-butyl, iso-butyl, tert-butyl,n-pentyl, isopentyl, neopentyl, n-hexyl, 3-methylhexyl,2,2-dimethylpentyl, 2,3-dimethylpentyl, n-heptyl, n-octyl, n-nonyl, andn-decyl.

The term “alkenyl” as used herein, means a straight or branchedhydrocarbon chain containing from 2 to 10 carbons and containing atleast one carbon-carbon double bond. The term “Cx-Cy alkyl” means astraight or branched hydrocarbon chain containing at least onecarbon-carbon double bond containing x to y carbon atoms. The term“C₂-C₄ alkenyl” means an alkenyl group containing 2-4 carbon atoms.Representative examples of alkenyl include, but are not limited tobuta-2,3-dienyl, ethenyl, 2-propenyl, 2-methyl-2-propenyl, 3-butenyl,4-pentenyl, 5-hexenyl, 2-heptenyl, 2-methyl-1-heptenyl, and 3-decenyl.

The term “alkenylene” means a divalent group derived from a straight orbranched chain hydrocarbon of 2 to 4 carbon atoms and contains at leastone carbon-carbon double bond. The term “Cx-Cy alkylene” means adivalent group derived from a straight or branched hydrocarbon chaincontaining at least one carbon-carbon double bond and containing x to ycarbon atoms. Representative examples of alkenylene include, but are notlimited to, —CH═CH— and —CH₂CH═CH—.

The term “alkynyl” as used herein, means a straight or branched chainhydrocarbon group containing from 2 to 10 carbon atoms and containing atleast one carbon-carbon triple bond. The term “Cx-Cy alkynyl” means astraight or branched chain hydrocarbon group containing from x to ycarbon atoms. Representative examples of alkynyl include, but are notlimited, to acetylenyl, 1-propynyl, 2-propynyl, 3-butynyl, 2-pentynyl,and 1-butynyl.

The term “alkynylene,” as used herein, means a divalent radical derivedfrom a straight or branched chain hydrocarbon group containing from 2 to10 carbon atoms and containing at least one carbon-carbon triple bond.

The term “aryl” (alone or in combination with another term(s)) means anaromatic carbocyclyl containing from 6 to 14 carbon ring atoms. An arylmay be monocyclic or polycyclic (i.e., may contain more than one ring).In the case of polycyclic aromatic rings, only one ring the polycyclicsystem is required to be unsaturated while the remaining ring(s) may besaturated, partially saturated or unsaturated. Examples of aryls includephenyl, naphthalenyl, indenyl, indanyl, and tetrahydronaphthyl.

The term “carbocyclyl” (alone or in combination with another term(s))means a saturated cyclic (i.e., “cycloalkyl”), partially saturatedcyclic (i.e., “cycloalkenyl”), or completely unsaturated (i.e., “aryl”)hydrocarbyl substituent containing from 3 to 14 carbon ring atoms (“ringatoms” are the atoms bound together to form the ring or rings of acyclic substituent). A carbocyclyl may be a single-ring (monocyclic) orpolycyclic ring structure.

A carbocyclyl may be a single ring structure, which typically containsfrom 3 to 8 ring atoms, more typically from 3 to 6 ring atoms, and evenmore typically 5 to 6 ring atoms. Examples of such single-ringcarbocyclyls include cyclopropyl (cyclopropanyl), cyclobutyl(cyclobutanyl), cyclopentyl (cyclopentanyl), cyclopentenyl,cyclopentadienyl, cyclohexyl (cyclohexanyl), cyclohexenyl,cyclohexadienyl, and phenyl. A carbocyclyl may alternatively bepolycyclic (i.e., may contain more than one ring). Examples ofpolycyclic carbocyclyls include bridged, fused, and spirocycliccarbocyclyls. In a spirocyclic carbocyclyl, one atom is common to twodifferent rings. An example of a spirocyclic carbocyclyl substituent isspirocyclopropyl. In a spirocyclic carbocyclyl, one atom is common totwo different rings. An example of a spirocyclic carbocyclyl isspiropentanyl. In a bridged carbocyclyl, the rings share at least twocommon non-adjacent atoms. Examples of bridged carbocyclyls includebicyclo[2.2.1]heptanyl, bicyclo[2.2.1]hept-2-enyl, and adamantanyl. In afused-ring carbocyclyl system, two or more rings may be fused together,such that two rings share one common bond. Examples of two- orthree-fused ring carbocyclyls include naphthalenyl,tetrahydronaphthalenyl (tetralinyl), indenyl, indanyl (dihydroindenyl),anthracenyl, phenanthrenyl, and decalinyl.

The term “cyclic moiety,” as used herein, means benzene, phenyl,phenylene, cycloalkane, cycloalkyl, cycloalkylene, cycloalkene,cycloalkenyl, cycloalkenylene, cycloalkyne, cycloalkynyl,cycloalkynylene, heteroarene, heteroaryl, heterocycloalkane,heterocycloalkyl, heterocycloalkene, heterocycloalkenyl and spiroalkyl.

The term “cycloalkylene” or cycloalkyl” or “cycloalkane” as used herein,means a monocyclic or bridged hydrocarbon ring system. The monocycliccycloalkyl is a carbocyclic ring system containing three to eight carbonatoms, zero heteroatoms and zero double bonds. Examples of monocyclicring systems include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cycloheptyl, and cyclooctyl. The monocyclic ring may contain one or twoalkylene bridges, each consisting of one, two, or three carbon atoms,each linking two non-adjacent carbon atoms of the ring system.Non-limiting examples of such bridged cycloalkyl ring systems includebicyclo[3.1.1]heptane, bicyclo[2.2.1]heptane, bicyclo[2.2.2]octane,bicyclo[3.2.2]nonane, bicyclo[3.3.1]nonane, bicyclo[4.2.1]nonane,tricyclo[3.3.1.0.sup.3,7]nonane (octahydro-2,5-methanopentalene ornoradamantane), and tricyclo[3.3.1.1.sup.3,7]decane (adamantane). Themonocyclic and bridged cycloalkyl can be attached to the parentmolecular moiety through any substitutable atom contained within thering system.

The term “cycloalkenylene,” or “cycloalkenyl” or “cycloalkene” as usedherein, means a monocyclic or a bridged hydrocarbon ring system. Themonocyclic cycloalkenyl has four-, five-, six-, seven- or eight carbonatoms and zero heteroatoms. The four-membered ring systems have onedouble bond, the five- or six-membered ring systems have one or twodouble bonds, and the seven- or eight-membered ring systems have one,two, or three double bonds. Representative examples of monocycliccycloalkenyl groups include, but are not limited to, cyclobutenyl,cyclopentenyl, cyclohexenyl, cycloheptenyl, and cyclooctenyl. Themonocyclic cycloalkenyl ring may contain one or two alkylene bridges,each consisting of one, two, or three carbon atoms, each linking twonon-adjacent carbon atoms of the ring system. Representative examples ofthe bicyclic cycloalkenyl groups include, but are not limited to,4,5,6,7-tetrahydro-3aH-indene, octahydronaphthalenyl, and1,6-dihydro-pentalene. The monocyclic and bicyclic cycloalkenyl can beattached to the parent molecular moiety through any substitutable atomcontained within the ring systems.

The term “cycloalkyne,” or “cycloalkynyl,” or “cycloalkynylene,” as usedherein, means a monocyclic or a bridged hydrocarbon ring system. Themonocyclic cycloalkynyl has eight or more carbon atoms, zeroheteroatoms, and one or more triple bonds. The monocyclic cycloalkynylring may contain one or two alkylene bridges, each consisting of one,two, or three carbon atoms, each linking two non-adjacent carbon atomsof the ring system. The monocyclic and bridged cycloalkynyl can beattached to the parent molecular moiety through any substitutable atomcontained within the ring systems.

The term “heteroarene,” or “heteroaryl,” or “heteroarylene,” as usedherein, means a five-membered or six-membered aromatic ring having atleast one carbon atom and one or more than one independently selectednitrogen, oxygen or sulfur atom. The heteroarenes of this disclosure areconnected through any adjacent atoms in the ring, provided that propervalences are maintained. Specifically, the term “heteroaryl” (alone orin combination with another term(s)) means an aromatic heterocyclylcontaining from 5 to 14 ring atoms. A heteroaryl may be a single ring or2 or 3 fused rings. Examples of heteroaryl substituents include6-membered ring substituents such as pyridyl, pyrazyl, pyrimidinyl,pyridazinyl, and 1,3,5-, 1,2,4- or 1,2,3-triazinyl; 5-membered ringsubstituents such as imidazyl, furanyl, thiophenyl, pyrazolyl, oxazolyl,isoxazolyl, thiazolyl, 1,2,3-, 1,2,4-, 1,2,5-, or 1,3,4-oxadiazolyl andisothiazolyl; 6/5-membered fused ring substituents such asbenzothiofuranyl, benzisoxazolyl, benzoxazolyl, imidazolyl, indolyl,benzoimidazolyl, pyrrolo[2,3-b]pyridinyl, purinyl, and anthranilyl; and6/6-membered fused rings such as benzopyranyl, quinolinyl,isoquinolinyl, cinnolinyl, quinazolinyl, and benzoxazinyl.

The term “heterocyclyl” (alone or in combination with another term(s))means a saturated (i.e., “heterocycloalkyl”), partially saturated (i.e.,“heterocycloalkenyl”), or completely unsaturated (i.e., “heteroaryl”)ring structure containing a total of 3 to 14 ring atoms. At least one ofthe ring atoms is a heteroatom (i.e., oxygen, nitrogen, or sulfur), withthe remaining ring atoms being independently selected from the groupconsisting of carbon, oxygen, nitrogen, and sulfur. A heterocyclyl maybe a single-ring (monocyclic) or polycyclic ring structure. Aheterocyclyl may be a single ring, which typically contains from 3 to 7ring atoms, more typically from 3 to 6 ring atoms, and even moretypically 5 to 6 ring atoms. Examples of single-ring heterocyclylsinclude furanyl, dihydrofuranyl, tetrahydrofuranyl, tetrahydropyranyl,thiophenyl (thiofuranyl), dihydrothiophenyl, tetrahydrothiophenyl,pyrrolyl, pyrrolinyl, pyrrolidinyl, imidazolyl, imidazolinyl,imidazolidinyl, pyrazolyl, pyrazolinyl, pyrazolidinyl, triazolyl,tetrazolyl, oxazolyl, oxazolidinyl, isoxazolidinyl, isoxazolyl,thiazolyl, isothiazolyl, thiazolinyl, isothiazolinyl, thiazolidinyl,isothiazolidinyl, thiodiazolyl, oxadiazolyl (including1,2,3-oxadiazolyl, 1,2,4-oxadiazolyl, 1,2,5-oxadiazolyl (furazanyl), or1,3,4-oxadiazolyl), oxatriazolyl (including 1,2,3,4-oxatriazolyl or1,2,3,5-oxatriazolyl), oxetanyl, dioxazolyl (including 1,2,3-dioxazolyl,1,2,4-dioxazolyl, 1,3,2-dioxazolyl, or 1,3,4-dioxazolyl), oxathiazolyl,oxathiolyl, oxathiolanyl, pyranyl, dihydropyranyl, thiopyranyl,tetrahydrothiopyranyl, pyridinyl (azinyl), piperidinyl, diazinyl(including pyridazinyl (1,2-diazinyl), pyrimidinyl (1,3-diazinyl), orpyrazinyl (1,4-diazinyl)), piperazinyl, triazinyl (including1,3,5-triazinyl, 1,2,4-triazinyl, and 1,2,3-triazinyl)), oxazinyl(including 1,2-oxazinyl, 1,3-oxazinyl, or 1,4-oxazinyl)), oxathiazinyl(including 1,2,3-oxathiazinyl, 1,2,4-oxathiazinyl, 1,2,5-oxathiazinyl,or 1,2,6-oxathiazinyl)), oxadiazinyl (including 1,2,3-oxadiazinyl,1,2,4-oxadiazinyl, 1,4,2-oxadiazinyl, or 1,3,5-oxadiazinyl)),morpholinyl, azepinyl, oxepinyl, thiepinyl, and diazepinyl.

A heterocyclyl may alternatively be polycyclic (i.e., may contain morethan one ring). Examples of polycyclic heterocyclyls include bridged,fused, and spirocyclic heterocyclyls. In a spirocyclic heterocyclyl, oneatom is common to two different rings. Examples of spirocyclicheterocyclyls include 2-oxaspiro[3.5]nonanyl. Examples of polycyclicheterocyclyls include bridged, fused, and spirocyclic heterocyclyls. Ina spirocyclic heterocyclyl, one atom is common to two different rings.Examples of spirocyclic heterocyclyl include 2-oxaspiro[3.5]nonanyl. Ina bridged heterocyclyl, the rings share at least two common non-adjacentatoms. In a fused-ring heterocyclyl, two or more rings may be fusedtogether, such that two rings share one common bond. Examples of fusedring heterocyclyls containing two or three rings include indolizinyl,pyranopyrrolyl, 4H-quinolizinyl, purinyl, naphthyridinyl,pyrrolo[2,3-b]pyridinyl, pyridopyridinyl (includingpyrido[3,4-b]-pyridinyl, pyrido[3,2-b]-pyridinyl, orpyrido[4,3-b]-pyridinyl), and pteridinyl. Other examples of fused-ringheterocyclyls include benzo-fused heterocyclyls, such as indolyl,indazoyl, isoindolyl (isobenzazolyl, pseudoisoindolyl), indoleninyl(pseudoindolyl), isoindazolyl (benzpyrazolyl), benzoimidazolyl,benzazinyl (including quinolinyl (1-benzazinyl) or isoquinolinyl(2-benzazinyl)), phthalazinyl, quinoxalinyl, quinazolinyl, benzodiazinyl(including cinnolinyl (1,2-benzodiazinyl) or quinazolinyl(1,3-benzodiazinyl)), benzopyranyl (including chromanyl orisochromanyl), benzoxazinyl (including 1,3,2-benzoxazinyl,1,4,2-benzoxazinyl, 2,3,1-benzoxazinyl, or 3,1,4-benzoxazinyl), andbenzisoxazinyl (including 1,2-benzisoxazinyl or 1,4-b enzi soxazinyl).

The term “heterocycloalkyl” (alone or in combination with anotherterm(s)) means a saturated heterocyclyl.

The term “heterocycloalkene,” or “heterocycloalkenyl,” or“heterocycloalkenylene,” as used herein, means monocyclic or bridgedthree-, four-, five-, six-, seven-, or eight-membered ring containing atleast one heteroatom independently selected from the group consisting of0, N, and S and one or more double bonds. The monocyclic and bridgedheterocycloalkene are connected to the parent molecular moiety throughany substitutable carbon atom or any substitutable nitrogen atomcontained within the rings. The nitrogen and sulfur heteroatoms in theheterocycle rings may optionally be oxidized and the nitrogen atoms mayoptionally be quarternized. Representative examples of heterocycloalkenegroups include, but are not limited to, tetrahydrooxocinyl,1,4,5,6-tetrahydropyridazinyl, 1,2,3,6-tetrahydropyridinyl,dihydropyranyl, imidazolinyl, isothiazolinyl, oxadiazolinyl,isoxazolinyl, oxazolinyl, pyranyl, pyrazolinyl, pyrrolinyl,thiadiazolinyl, thiazolinyl, and thiopyranyl.

The term “phenylene,” as used herein, means a divalent radical formed byremoval of a hydrogen atom from phenyl.

The term “spiroalkyl,” as used herein, means alkylene, both ends ofwhich are attached to the same carbon atom and is exemplified byC₂-spiroalkyl, C₃-spiroalkyl, C₄-spiroalkyl, C₅-spiroalkyl,C₆-spiroalkyl, C₇-spiroalkyl, C₈-spiroalkyl, C₉-spiroalkyl and the like.

The term “spiroheteroalkyl,” as used herein, means spiroalkyl having oneor two CH.sub.2 moieties replaced with independently selected O, C(O),CNOH, CNOCH₃, S, S(O), SO₂ or NH and one or two CH moieties unreplacedor replaced with N.

The term “spiroheteroalkenyl,” as used herein, means spiroalkenyl havingone or two CH.sub.2 moieties replaced with independently selected O,C(O), CNOH, CNOCH₃, S, S(O), SO₂ or NH and one or two CH moietiesunreplaced or replaced with N and also means spiroalkenyl having one ortwo CH.sub.2 moieties unreplaced or replaced with independently selectedO, C(O), CNOH, CNOCH₃, S, S(O), SO₂ or NH and one or two CH moietiesreplaced with N.

The term, “spirocyclo,” as used herein, means two substituents on thesame carbon atom that, together with the carbon atom to which they areattached, form a cycloalkane, heterocycloalkane, cycloalkene, orheterocycloalkene ring.

As stated, the selective Bcl-2 inhibitor compounds of the currentdisclosure encompass all possible combinations of the substituents forthe genus compound of the formulas. Suitable examples of compounds thatfall within the scope of the current disclosure include, but are notlimited to,

-   N-({5-chloro-6-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-[(6-fluoro-1H-indazol-4-yl)oxy]benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-{[4-({[(2S)-4-cyclopropylmorpholin-2-yl]methyl}amino)-3-nitrophenyl]sulfonyl}-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   N-({5-chloro-6-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-[(6-fluoro-1H-indol-5-yl)oxy]-N-({4-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]-3-nitrophenyl}sulfonyl)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(4,4-difluorocyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   2-(1H-benzimidazol-4-yloxy)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({5-fluoro-6-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]pyridin-3-yl}sulfonyl)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfony-1)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   N-({3-chloro-4-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]phenyl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   2-(1H-benzimidazol-4-yloxy)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(4-cyanocyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]benzamide;-   N-({5-chloro-6-[(cis-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   N-[(3-chloro-4-{[4-fluoro-1-(oxetan-3-yl)piperidin-4-yl]methoxy}phenyl)sulfonyl]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({5-cyano-6-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]pyridin-3-yl}sulfonyl)-2-(1H-indol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(4-fluorotetrahydro-2H-pyran-4-yl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   N-({3-chloro-4-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]phenyl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({5-fluoro-6-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]pyridin-3-yl}sulfonyl)-2-(1H-indazol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-{[4-({[(2R)-4-cyclopropylmorpholin-2-yl]methyl}amino)-3-nitrophenyl]sulfonyl}-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(trans-4-cyanocyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   Trans-2-[(6-amino-5-chloropyridin-3-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({4-[(4-morpholin-4-ylcyclohexyl)amino]-3-nitrophenyl}sulfonyl)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-{[4-({(3R)-1-[2-fluoro-1-(fluoromethyl)ethyl]pyrrolidin-3-yl}amino)-3-nitrophenyl]sulfonyl}-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   Trans-N-({5-chloro-6-[(4-hydroxycyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   N-({3-chloro-4-[(trans-4-hydroxycyclohexyl)methoxy]phenyl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   N-({5-chloro-6-[(trans-4-hydroxycyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-[(6-fluoro-1H-indazol-4-yl)oxy]benzamide;-   2-[(6-amino-5-chloropyridin-3-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[trans-4-(morpholin-4-yl)cyclohexyl]amino}-3-nitrophenyl)sulfonyl]benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(cis-4-hydroxy-4-methyl    cyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({5-cyano-6-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]pyridin-3-yl}sulfonyl)-2-(1H-indazol-4-yloxy)benzamide;-   N-[(5-chloro-6-{[4-fluoro-1-(oxetan-3-yl)piperidin-4-yl]methoxy}pyridin-3-yl)sulfonyl]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   2-[(6-amino-5-chloropyridin-3-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({4-[(4-methylpiperazin-1-yl)amino]-3-nitrophenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   Trans-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(4-methoxycyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   Trans-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({4-[(4-morpholin-4-ylcyclohexyl)amino]-3-nitrophenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({4-[(4-fluorotetrahydro-2H-pyran-4-yl)methoxy]-3-nitrophenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   2-[(6-amino-5-chloropyridin-3-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(3R)-1-(2,2-difluoroethyl)pyrrolidin-3-yl]amino}-3-nitrophenyl)sulfonyl]benzamide;-   N-({5-chloro-6-[(trans-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   N-({5-chloro-6-[(cis-1-fluoro-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   2-[(6-amino-5-chloropyridin-3-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-[(4-methoxycyclohexyl)methyl]amino-3-nitrophenyl)sulfonyl]benzamide;-   N-({5-chloro-6-[(trans-1-fluoro-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indazol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(trans-4-hydroxy-4-methyl    cyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   2-[(3-amino-1H-indazol-4-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(trans-4-methoxycyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({3-nitro-4-[(2-oxaspiro[3.5]non-7-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({5-cyano-6-[(trans-4-hydroxy-4-methylcyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-2-(1H-indazol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-[(6-fluoro-1H-indol-5-yl)oxy]-N-{[3-nitro-4-({[4-(oxetan-3-yl)morpholin-2-yl]methyl}amino)phenyl]sulfonyl}benzamide;-   N-({5-chloro-6-[(trans-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-[(6-fluoro-1H-indazol-4-yl)oxy]benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(5-cyano-6-{[4-fluoro-1-(oxetan-3-yl)piperidin-4-yl]methoxy}pyridin-3-yl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   2-[(6-amino-5-chloropyridin-3-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-[(4-hydroxycyclohexyl)methyl]amino-3-nitrophenyl)sulfonyl]benzamide;-   N-({5-chloro-6-[(trans-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-2-[(3-chloro-1H-indazol-4-yl)oxy]-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)benzamide;-   4-[4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}(²H₈)piperazin-1-yl]-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   N-({5-chloro-6-[(trans-1-fluoro-4-hydroxy-4-methyl    cyclohexyl)methoxy]pyridin-3-yl}sulfonyl)-4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(6-{[(cis-4-hydroxy-4-methyl    cyclohexyl)methyl]amino}-5-nitropyridin-3-yl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-(5-nitro-6-{[(tetrahydro-2H-pyran-4-ylmethyl)amino]pyridin-3-yl}sulfonyl)-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-({6-[(trans-4-hydroxy-4-methyl    cyclohexyl)methoxy]-5-(trifluoromethyl)pyridin-3-yl}sulfonyl)-2-(1H-indazol-4-yloxy)benzamide;-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-N-[(4-{[(cis-4-ethyl-4-hydroxycyclohexyl)methyl]amino}-3-nitrophenyl)sulfonyl]-2-(1H-pyrrolo[2,3-b]pyridin-5-yloxy)benzamide;    and-   4-(4-{[2-(4-chlorophenyl)-4,4-dimethylcyclohex-1-en-1-yl]methyl}piperazin-1-yl)-2-(1H-indol-5-yloxy)-N-({3-nitro-4-[(tetrahydro-2H-pyran-4-ylmethyl)amino]phenyl}sulfonyl)benzamide.

Formulations

Further, the methods of the disclosure include administering compoundshaving the chemical names or formula reported herein with or without anexcipient. Excipients include, for example, encapsulating materials oradditives such as absorption accelerators, antioxidants, binders,buffers, coating agents, coloring agents, diluents, disintegratingagents, emulsifiers, extenders, fillers, flavoring agents, humectants,lubricants, perfumes, preservatives, propellants, releasing agents,sterilizing agents, sweeteners, solubilizers, wetting agents andmixtures thereof.

Excipients for preparation of compositions comprising a compound havingthe formula to be administered orally in solid dosage form include, forexample, agar, alginic acid, aluminum hydroxide, benzyl alcohol, benzylbenzoate, 1,3-butylene glycol, carbomers, castor oil, cellulose,cellulose acetate, cocoa butter, corn starch, corn oil, cottonseed oil,cross-povidone, diglycerides, ethanol, ethyl cellulose, ethyl laureate,ethyl oleate, fatty acid esters, gelatin, germ oil, glucose, glycerol,groundnut oil, hydroxypropylmethyl cellulose, isopropanol, isotonicsaline, lactose, magnesium hydroxide, magnesium stearate, malt,mannitol, monoglycerides, olive oil, peanut oil, potassium phosphatesalts, potato starch, povidone, propylene glycol, Ringer's solution,safflower oil, sesame oil, sodium carboxymethyl cellulose, sodiumphosphate salts, sodium lauryl sulfate, sodium sorbitol, soybean oil,stearic acids, stearyl fumarate, sucrose, surfactants, talc, tragacanth,tetrahydrofurfuryl alcohol, triglycerides, water, and mixtures thereof.Excipients for preparation of compositions comprising a compound of thisdisclosure having the formula to be administered ophthalmically ororally in liquid dosage forms include, for example, 1,3-butylene glycol,castor oil, corn oil, cottonseed oil, ethanol, fatty acid esters ofsorbitan, germ oil, groundnut oil, glycerol, isopropanol, olive oil,polyethylene glycols, propylene glycol, sesame oil, water and mixturesthereof. Excipients for preparation of compositions comprising acompound of this disclosure having the formula to be administeredosmotically include, for example, chlorofluorohydrocarbons, ethanol,water and mixtures thereof. Excipients for preparation of compositionscomprising a compound of this disclosure having the formula to beadministered parenterally include, for example, 1,3-butanediol, castoroil, corn oil, cottonseed oil, dextrose, germ oil, groundnut oil,liposomes, oleic acid, olive oil, peanut oil, Ringer's solution,safflower oil, sesame oil, soybean oil, U.S.P. or isotonic sodiumchloride solution, water and mixtures thereof. Excipients forpreparation of compositions comprising a compound of this disclosurehaving the formula to be administered rectally or vaginally include, forexample, cocoa butter, polyethylene glycol, wax and mixtures thereof.

The compounds used in the methods of the current disclosure may alsoinclude a pharmaceutically acceptable salt form of a compound having theformula. The phrase “pharmaceutically acceptable salt(s)”, as usedherein, means those salts of the selective Bcl-2 inhibitors of thedisclosure that are safe and effective for administration to a patientand that do not adversely affect the therapeutic qualities of thecompound. Pharmaceutically acceptable salts include salts of acidic orbasic groups present in compounds of the disclosure. Pharmaceuticallyacceptable acid addition salts include, but are not limited to,hydrochloride, hydrobromide, hydroiodide, nitrate, sulfate, bisulfate,phosphate, acid phosphate, isonicotinate, acetate, lactate, salicylate,citrate, tartrate, pantothenate, bitartrate, ascorbate, succinate,maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate,formate, benzoate, glutamate, methanesulfonate, ethanesulfonate,benzensulfonate, p-toluenesulfonate and pamoate (i.e.,1,1′-methylene-bis-(2-hydroxy-3-naphthoate)) salts. Certain compounds ofthe disclosure can form pharmaceutically acceptable salts with variousamino acids. Suitable base salts include, but are not limited to,aluminum, calcium, lithium, magnesium, potassium, sodium, zinc, anddiethanolamine salts. For a review on pharmaceutically acceptable saltssee Berge et al., 66 J. Pharm. Sci., 1-19 (1977), incorporated herein byreference, in its entirety.

The compounds used in the methods of the current disclosure may alsocomprise geometric isomers. Compounds of this disclosure may containcarbon-carbon double bonds or carbon-nitrogen double bonds in the E or Zconfiguration, wherein the term “E” represents higher order substituents on opposite sides of the carbon-carbon or carbon-nitrogendouble bond and the term “Z” represents higher order substituents on thesame side of the carbon-carbon or carbon-nitrogen double bond asdetermined by the Cahn-Ingold-Prelog Priority Rules. The compounds ofthis disclosure may also exist as a mixture of “E” and “Z” isomers.Substituents around a cycloalkyl or heterocycloalkyl are designated asbeing of cis or trans configuration. Furthermore, the disclosurecontemplates the various isomers and mixtures thereof resulting from thedisposal of sub stituents around an adamantane ring system. Twosubstituents around a single ring within an adamantane ring system aredesignated as being of Z or E relative configuration. For examples, seeC. D. Jones, M. Kaselj, R. N. Salvatore, W. J. le Noble J. Org. Chem.1998, 63, 2758-2760 and E. L. Eliel, and S. H. Wilen. (1994)Stereochemistry of Organic Compounds. New York, N.Y.: John Wiley & Sons,Inc.

The compounds may also contain asymmetrically substituted carbon atomsin the R or S configuration, in which the terms “R” and “S” are asdefined by the IUPAC 1974 Recommendations for Section E, FundamentalStereochemistry, Pure Appl. Chem. (1976) 45, 13-10. Compounds havingasymmetrically substituted carbon atoms with equal amounts of R and Sconfigurations are racemic at those carbon atoms. Atoms with an excessof one configuration over the other are assigned the configurationpresent in the higher amount, such an excess of about 85%-90%, an excessof about 95%-99%, or an excess greater than about 99%. Accordingly, thisdisclosure includes racemic mixtures, relative and absolutestereoisomers, and mixtures of relative and absolute stereoisomers.

The compounds used in the methods of the current disclosure containingNH, C(O)OH, OH or SH moieties may have attached thereto prodrug-formingmoieties. The prodrug-forming moieties are removed by metabolicprocesses and release the compounds having the freed hydroxyl, amino orcarboxylic acid in vivo. Prodrugs are useful for adjusting suchpharmacokinetic properties of the compounds as solubility and/orhydrophobicity, absorption in the gastrointestinal tract,bioavailability, tissue penetration, and rate of clearance.

The compounds used in the various embodiments can also exist inisotope-labeled or isotope-enriched form containing one or more atomshaving an atomic mass or mass number different from the atomic mass ormass number most abundantly found in nature. Isotopes can be radioactiveor non-radioactive isotopes. Isotopes of atoms such as hydrogen, carbon,phosphorous, sulfur, fluorine, chlorine, and iodine include, but are notlimited to, ²H, ³H, ¹³C, ¹⁴C, ¹⁵N, ¹⁸O, ³²P, ³⁵S, ¹⁸F, ³⁶Cl, and ¹²⁵ICompounds that contain other isotopes of these and/or other atoms arewithin the scope of this disclosure.

In another embodiment, the isotope-labeled compounds contain deuterium(²H), tritium (³H) or ¹⁴C isotopes. Isotope-labeled compounds of thisdisclosure can be prepared by the general methods well known to personshaving ordinary skill in the art. Such isotope-labeled compounds can beconveniently prepared by carrying out the procedures disclosed in theExamples disclosed herein and Schemes by substituting a readilyavailable isotope-labeled reagent for a non-labeled reagent. In someinstances, compounds may be treated with isotope-labeled reagents toexchange a normal atom with its isotope, for example, hydrogen fordeuterium can be exchanged by the action of a deuteric acid such asD₂SO₄/D₂O. In addition to the above, relevant procedures andintermediates are disclosed, for instance, in Lizondo, J et al., DrugsFut, 21(11), 1116 (1996); Brickner, S J et al., J Med Chem, 39(3), 673(1996); Mallesham, B et al., Org Lett, 5(7), 963 (2003); PCTpublications WO1997010223, WO2005099353, WO1995007271, WO2006008754;U.S. Pat. Nos. 7,538,189; 7,534,814; 7,531,685; 7,528,131; 7,521,421;7,514,068; 7,511,013; and US Patent Application Publication Nos.20090137457; 20090131485; 20090131363; 20090118238; 20090111840;20090105338; 20090105307; 20090105147; 20090093422; 20090088416; and20090082471, the methods are hereby incorporated by reference.

The isotope-labeled compounds of the disclosure may be used as standardsto determine the effectiveness of Bcl-2 inhibitors in binding assays.Isotope containing compounds have been used in pharmaceutical researchto investigate the in vivo metabolic fate of the compounds by evaluationof the mechanism of action and metabolic pathway of thenonisotope-labeled parent compound (Blake et al. J. Pharm. Sci. 64, 3,367-391 (1975)). Such metabolic studies are important in the design ofsafe, effective therapeutic drugs, either because the in vivo activecompound administered to the patient or because the metabolites producedfrom the parent compound prove to be toxic or carcinogenic (Foster etal., Advances in Drug Research Vol. 14, pp. 2-36, Academic press,London, 1985; Kato et al., J. Labelled Comp. Radiopharmaceut.,36(10):927-932 (1995); Kushner et al., Can. J. Physiol. Pharmacol., 77,79-88 (1999).

In addition, non-radioactive isotope containing drugs, such asdeuterated drugs called “heavy drugs,” can be used for the treatment ofdiseases and conditions related to Bcl-2 activity. Increasing the amountof an isotope present in a compound above its natural abundance iscalled enrichment. Examples of the amount of enrichment include fromabout 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 16, 21, 25, 29, 33, 37,42, 46, 50, 54, 58, 63, 67, 71, 75, 79, 84, 88, 92, 96, to about 100 mol%. Replacement of up to about 15% of normal atom with a heavy isotopehas been effected and maintained for a period of days to weeks inmammals, including rodents and dogs, with minimal observed adverseeffects (Czajka D M and Finkel A J, Ann. N.Y. Acad. Sci. 1960 84: 770;Thomson J F, Ann. New York Acad. Sci 1960 84: 736; Czakj a D M et al.,Am. J. Physiol. 1961 201: 357). Acute replacement of as high as 15%-23%in human fluids with deuterium was found not to cause toxicity(Blagojevic N et al. in “Dosimetry & Treatment Planning for NeutronCapture Therapy”, Zamenhof R, Solares G and Harling 0 Eds. 1994.Advanced Medical Publishing, Madison Wis. pp. 125-134; Diabetes Metab.23: 251 (1997)).

Stable isotope labeling of a drug can alter its physico-chemicalproperties such as pKa and lipid solubility. These effects andalterations can affect the pharmacodynamic response of the drug moleculeif the isotopic substitution affects a region involved in aligand-receptor interaction. While some of the physical properties of astable isotope-labeled molecule are different from those of theunlabeled one, the chemical and biological properties are the same, withone important exception: because of the increased mass of the heavyisotope, any bond involving the heavy isotope and another atom will bestronger than the same bond between the light isotope and that atom.Accordingly, the incorporation of an isotope at a site of metabolism orenzymatic transformation will slow said reactions potentially alteringthe pharmacokinetic profile or efficacy relative to the non-isotopiccompound.

The current methods may also incorporate a prodrug form of the selectivea compound disclosed herein. Prodrugs are derivatives of an active drugdesigned to ameliorate some identified, undesirable physical orbiological property. The physical properties are usually solubility (toomuch or not enough lipid or aqueous solubility) or stability related,while problematic biological properties include too rapid metabolism orpoor bioavailability which itself may be related to a physicochemicalproperty. Prodrugs are usually prepared by: a) formation of ester, hemiesters, carbonate esters, nitrate esters, amides, hydroxamic acids,carbamates, imines, Mannich bases, phosphates, phosphate esters, andenamines of the active drug, b) functionalizing the drug with azo,glycoside, peptide, and ether functional groups, c) use of aminals,hemi-aminals, polymers, salts, complexes, phosphoramides, acetals,hemiacetals, and ketal forms of the drug. For example, see AndrejusKorolkovas's, “Essentials of Medicinal Chemistry”, JohnWiley-Interscience Publications, John Wiley and Sons, New York (1988),pp. 97-118, which is incorporated in its entirety by reference herein.

Furthermore, the methods of the current disclosure may involveadministration of the compounds having The formula by, for example, atleast one mode selected from parenteral, subcutaneous, intramuscular,intravenous, intraarticular, intrabronchial, intraabdominal,intracapsular, intracartilaginous, intracavitary, intracelial,intracerebellar, intracerebroventricular, intracolic, intracervical,intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic,intrapericardiac, intraperitoneal, intrapleural, intraprostatic,intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal,intrasynovial, intrathoracic, intrauterine, intravesical, bolus,vaginal, rectal, buccal, sublingual, intranasal, and transdermal.

As previously stated, the “therapeutically effective amount” of thecurrent disclosure refers to that amount of the compound beingadministered sufficient to prevent development of or alleviate to someextent one or more of the symptoms of the condition or disorder beingtreated. Therapeutically effective amounts of compounds having theformula depend on the recipient of the treatment, the disorder beingtreated and the severity thereof, the composition containing thecompound, the time of administration, the route of administration, theduration of treatment, the compound potency, its rate of clearance andwhether or not another drug is co-administered. Generally, the methodsof the current disclosure involve administering a dose of the selectiveBcl-2 inhibitor ranging from about 0.001 mg/kg to about 1000 mg/kg. Inone embodiment, the methods involve administering a dose of selectiveBcl-2 inhibitor ranging from about 0.01 mg/kg to about 500 mg/kg. In afurther embodiment, the methods involve administering a dose ofselective Bcl-2 inhibitor ranging from about 0.1 mg/kg to about 300mg/kg.

EXAMPLES

Reduced SDH activity in ABT-199 sensitive MM

SDH/respiratory Complex II is an enzyme that has a direct role both inthe TCA cycle and electron transport chain and consequently OCR andoxidative phosphorylation. Complex II activity was evaluated in ABT-199sensitive and resistant cells. SDH consists of 4 subunits: SDHA (FADbinding site), SDHB (Fe-S containing subunit), SDHC and SDHD (containinga heme band ubiquinone binding Qp site). Electrons released from SDHAupon the oxidation of succinate to fumarate are transferred sequentiallyfrom FAD to Fe—S clusters to ubiquinone at the Qp site. The reduction ofubiquinone bound in the Qp site to ubiquinol is referred to as theubiquinone reductase activity of SDH. The reduction of ubiquinonefacilitates the extrusion of protons from Complex Ill facilitating ATPsynthesis. SQR activity is measured in live permeabilized cells, withintact mitochondria by pre-treating with inhibitors of Complex I(rotenone) and Complex III (antimycin) leaving Complex II/SDH activityintact. SQR activity correlates with electron transfer from ubiquinonebound in SQR's Qp site to the redox sensitive dye, DCIPIP. The assay isvalidated by detection of SDH activity only upon addition of thesubstrate succinate and lack of activity upon addition of malonate (SDHAinhibitor) or SQR inhibitor thenoyltrifluoroacetone (TTFA) (FIG. 1A).ABT-199 sensitive cells exhibit low SQR activity while resistant linesincluding t(11; 14) U266 exhibit high SQR activity (FIG. 1B),importantly correlating with ABT-199 sensitivity.

Inhibition of SQR with Qp Site Inhibitor Thenoyltrifluoroacetone (TTFA)Sensitizes Resistant Cells to ABT-199

Reduced SQR activity in ABT-199 sensitive cells prompted testing todetermine whether inhibition of SQR with the Qp site inhibitor TTFAsensitizes resistant MM to ABT-199. The maximal dose of TTFA that didnot cause cytotoxicity in MM was tested. This amount inhibited SQRactivity. Resistant MM lines were treated with TTFA and/or a dose rangeof ABT-199 and cell death evaluated by Annexin V/DAPI staining.Inhibition of SQR induced potent sensitization to ABT-199 in resistantlines and MM patient cells (FIG. 2A). Importantly, patient sample #3 wasa t(11;14) ABT-199 sensitive sample that had low SQR activity confirmingour hypothesis that reduced SQR activity correlates with ABT-199sensitivity. Inhibition of Complex I or Complex III with 3NPA (whichtargets the SOHA subunit) at the maximum dose that did not elicitcytotoxicity, were not as effective as TTFA in inducing sensitization toABT-19 underscoring the importance of targeting the Qp site foreffective sensitization to ABT-199. TTFA or ABT-199 individually did notelicit cytotoxicity in MM. Importantly; normal cells contained within amyeloma patient sample did not exhibit significant cytotoxicity upontreatment with TTFA and ABT-199 (FIG. 2B) supporting selectivecytotoxicity in tumor cells.

1. A method of treating cancer comprising administering an effectiveamount of a Bcl-2 inhibitor in combination with a mitochondrial complexII inhibitor to a subject in need thereof.
 2. The method of claim 1,wherein the mitochondrial complex II inhibitor isthenoyltrifluoroacetone or atpenin A5.
 3. The method of claim 1, whereinthe Bcl-2 inhibitor is venetoclax or navitoclax.
 4. The method of claim1 wherein the Bcl-2 inhibitor has the following formula:

or pharmaceutically acceptable salt thereof, wherein A¹ is C(A²); A² isH, F, Br, I, or Cl; B¹ is R¹, NHR¹, NHC(O)R¹, F, Br, I, or Cl; D¹ is H,F, Br, I, or Cl; E¹ is H; and Y¹ is H, CN, NO₂, F, Cl, Br, I, CF₃, R¹⁷,OR¹⁷, SR¹⁷, SO₂R¹⁷, or C(O)NH₂; R¹ is R⁴ or R⁵; R⁴ is cycloalkyl orheterocycloalkyl; R⁵ is alkyl or alkynyl, each of which is unsubstitutedor substituted with one or two or three substituents independentlyselected from the group consisting of R⁷, OR⁷, NHR⁷, N(R⁷)₂, CN, OH, F,Cl, Br, and I; R⁷ is R⁸, R⁹, R¹⁰, or R¹¹; R⁸ is phenyl; R⁹ isheteroaryl; R¹⁰ is cycloalkyl, cycloalkenyl, or heterocycloalkyl; eachof which is unfused or fused with R^(10A); R^(10A) is heteroarene; R¹¹is alkyl, which is unsubstituted or substituted with one or two or threesubstituents independently selected from the group consisting of R¹²,OR¹², and CF₃; R¹² is R¹⁴ or R¹⁶; R¹⁴ is heteroaryl; R¹⁶ is alkyl; R¹⁷is alkyl or alkynyl, each of which is unsubstituted or substituted withone or two or three substituents independently selected from the groupconsisting of R²², F, Cl, Br and I; R²² is heterocycloalkyl; wherein thecyclic moieties represented by R⁴, R⁸, R¹⁰, R²², are independentlyunsubstituted or substituted with one or two or three or four or fivesubstituents independently selected from the group consisting ofR^(57A), R²⁷, OR⁵⁷, SO₂R⁵⁷, C(O)R⁵⁷, C(O)OR⁵⁷, C(O)N(R⁵⁷)₂, NH₂, NHR⁵⁷,N(R⁵⁷)₂, NHC(O)R⁵⁷, NHS(O)₂R⁵⁷, OH, CN, (O), F, Cl, Br and I; R^(57A) isspiroalkyl or spiroheteroalkyl; R⁵⁷ is R⁵⁸, R⁶⁰, or R⁶¹; R⁵⁸ is phenyl;R⁶⁰ is cycloalkyl or heterocycloalkyl; R⁶¹ is alkyl, which isunsubstituted or substituted with one or two or three substituentsindependently selected from the group consisting of R⁶², OR⁶², N(R⁶²)₂,C(O)OH, CN, F, Cl, Br, and I; R⁶² is R⁶⁵ or R⁶⁶; R⁶⁵ is cycloalkyl orheterocycloalkyl; R⁶⁶ is alkyl, which is unsubstituted or substitutedwith OR⁶⁷; R⁶⁷ is alkyl; wherein the cyclic moieties represented byR^(57A), R⁵⁸, and R⁶⁰ are unsubstituted or substituted with one or twoor three or four substituents independently selected from the groupconsisting of R⁶⁸, F, Cl, Br, and I; R⁶⁸ is R⁷¹ or R⁷²; R⁷¹ isheterocycloalkyl; and R⁷² is alkyl, which is unsubstituted orsubstituted with one or two F.
 5. The method of claim 1, the Bcl-2inhibitor has the following formula:

or pharmaceutically acceptable salts thereof wherein, A¹ is N or CH; B¹is OR¹ or NHR¹; Y¹ is CN, NO₂, CF₃, F or Cl; R¹ is (CH₂)_(n)R²; R² iscycloalkyl or heterocyclyl; wherein the heterocyclyl and cycloalkyl areoptionally substituted with one or more independently selected R⁴, OR⁴,OH, CN, or F; R³ is heteroaryl; wherein the heteroaryl is optionallysubstituted with one or more independently selected NH₂, C₁, or F; R⁴ isalkyl, cycloalkyl, heterocyclyl, or spiroheterocyclyl; wherein the alkylis optionally substituted with one or more F; R⁵ is hydrogen ordeuterium; each R⁶ is independently selected from CH₃, spirocyclopropyland OH; m is 0, 1, 2, 3, 4, 5, or 6; n is 0 or 1; and p is 0, 1, or 2.6. The method of claim 1, wherein the cancer is a hematologicalmalignancy selected from multiple myeloma, leukemia, or lymphoma.
 7. Themethod of claim 1, wherein the hematological malignancy is acutelymphoblastic leukemia (ALL), acute myelogenous leukemia (AML), chroniclymphocytic leukemia (CLL), small lymphocytic lymphoma (SLL), chronicmyelogenous leukemia, acute monocytic leukemia (AMOL), Hodgkin'slymphomas, and non-Hodgkin's lymphomas such as Burkitt lymphoma, B-celllymphoma.
 8. The method of claim 1, wherein the subject is diagnosedwith cancer or hematological malignancy.
 9. The method of claim 1wherein administering an effective amount of a Bcl-2 inhibitor incombination with a mitochondrial complex II inhibitor is further incombination with an additional chemotherapy agent.
 10. A method oftreating multiple myeloma comprising administering an effective amountof venetoclax in combination with a thenoyltrifluoroacetone or atpeninA5 to a subject in need thereof.
 11. A method of comprising, a)isolating a sample of cancer cells from a subject, b) mixing the samplewith mitochondrial complex II, succinate, and ubiquinone; and c)measuring succinate ubiquinone reductase (SQR) activity providingmeasured SQR activity.
 12. The method of claim 11 further comprisingdiagnosing the subject as sensitive to or in need of a chemotherapytreatment comprising administering a Bcl-2 inhibitor in combination witha mitochondrial complex II inhibitor, if the measured SQR activity ishigher than a control level.
 13. The method of claim 11 furthercomprising diagnosing the subject as not in need of or would not benefitfrom a chemotherapy comprising administering Bcl-2 inhibitor incombination with a mitochondrial complex II inhibitor, if the measuredSQR activity is lower than a control level.
 14. The method of claim 11,further comprising the step of recording the measurements.
 15. Themethod of claim 14, wherein the measurements are recorded in anelectronic format.
 16. The method of claim 12, further comprising thestep of recording the diagnosis.
 17. The method of claim 16, wherein thediagnosis is recorded in an electronic format.
 18. The method of claim17, further comprising the step of reporting the measurements ordiagnosis to a medical professional, the subject, or representativethereof.
 19. The method of claim 12, further comprising administering aneffective amount of a combination therapy of Bcl-2 inhibitor incombination with a mitochondrial complex II inhibitor to the subject.20. A method of diagnosing and treating multiple myeloma comprising, a)isolating a sample of myeloma cells from a subject, b) mixing the samplewith mitochondrial complex II, succinate, and ubiquinone; and c)measuring succinate ubiquinone reductase (SQR) activity providingmeasured SQR activity; and d) treating the subject with venetoclax incombination with thenoyltrifluoroacetone or atpenin A5 if the measuredSQR activity is higher than a control level.